CN1990462A - Use of succinyl hydrazine compounds and compositions thereof in preparation of drug for treating leukemia - Google Patents

Abstract

The invention relates to succinyl hydrazine compounds derivative and medical compound containing them; and the application of it in preparing medicine that resists leukemia. The succinyl hydrazine compounds derivative shows inhibiting action to BCR-Ab1 tyrosine kinase activity through pharmacological test. It is characterized by high effecivity and low toxicity, and can be widly used to prepare medicine that treats chronic granulocytic leukemia (CML) and philadelphia chromosome positive acute lymphoblastic leukemia, especially medicine treating patient that generates drug resistance to Gleevec.

Description

The application aspect preparation treatment anti-leukemia medicine of succinhydrazide compounds and composition thereof

Technical field

The invention belongs to medical technical field, or rather, relate to a kind of succinhydrazide compounds and preparation method thereof with anti-tumor activity, and the application of composition aspect preparation treatment chronic myelocytic leukemia (CML) and Philadelphia chromosome male acute lymphoblastic leukemia (ALL) medicine that contains the succinhydrazide compounds.

Background technology

There are Ph karyomit(e): t (9 in 95% chronic myelogenous leukemia (CML) and 10 ~ 25% adult's acute lymphoblastic leukemias (ALL); 22) (q34; Q11).BCR district (breaking point polymeric area) on proto-oncogene c-ABL transposition to 22 karyomit(e) on No. 9 karyomit(e) produces the BCR-ABL mosaic gene, and expresses BCR-ABL protein.This albumen is compared with normal abl gene albumen, and tyrosine kinase activity causes malignant transformation of cells because of losing regulation and control.At the CML chronic phase, can control by cell toxicity medicament (hydroxyurea, Myelosan etc.), in case the final sudden turn of events of disease, the patient is because of out of hand and dead; The application of interferon alpha can make patient P h karyomit(e) turn out cloudy clinically in recent years, but the effect of raising patient chance for survival is still imprecise.Therefore, seeking new methods of treatment, to reach the leukemic purpose of radical cure imperative.In forming, CML and part A LL play critical biological action according to Tyrosylprotein kinase, the treatment of designing at the tyrosine kinase activity of BCR-ABL fusion rotein is proved to be a kind of effective therapeutic strategy, has become the main direction of research in recent years.The treatment that appears as CML of Gleevec has brought hope.

Gleevec has another name called imatinib (imatinib, STI571), chemistry 4-[(4-methyl isophthalic acid-piperazinyl by name) methyl]-N-[4-methyl-3-[[4-(3-pyridyl)-2-pyrimidyl] amino] phenyl] the benzamide methane sulfonates, it is a novel tyrosine kinase inhibitor, be first molecular targeted property medicine at oncogene, act on target ABL, BCR/ABL, thrombocyte differential growth factor acceptor (platelet-derived growth factor receptor, PDGFR) and multiple Tyrosylprotein kinase such as TEL-PDGFR.Gleevec is the specific small-molecule drug that causes tumorigenic genetic flaw of first target that obtains the drugs approved by FDA listing, as special efficacy " intelligent bomb ", by combining of competitive inhibition ABL and BCR/ABL kinases and ATP, the activity that suppresses these Tyrosylprotein kinases, the afunction that causes BCR/ABL, thereby the propagation of anticancer.Therefore, suppress generation and development that the BCR/ABL fusion rotein just can stop CML, and performance treatment effect is chosen as one of big technological breakthrough of calendar year 2001s ten by " Science " magazine.In clinical trial, oral once a day Gleevec, dosage is increased to 1000mg gradually from 25mg, does not find dose limitation toxicity.In the dosage group of 300mg-1000mg, the 54 examples previously invalid CML chronic phase patients of interferon therapy all obtain hematologic response, efficiently reach 100%, and 98% (53 example) reaches fully and alleviate; Wherein still cytogenetics alleviation of 53% (29 example).This medicine has been obtained good curative effect at clinical treatment CML and part A LL, and Gleevec also blocks other molecular pathway (comprising lung, prostate gland and brain) that participates in some entity tumor growth.And Gleevec is unlike chemotherapeutic agent or radiotherapy, also can injuring normal cell except destroying cancer cells.The discovery of Gleevec opened up with molecular biology theoretical suppress the conduction of oncogene signal control cancer novel method, the New Times of having started cancer therapy.

Yet the patient need take Gleevec continuously to keep drug effect, and some patients are recurrence after the treatment several months, and are no longer responsive to this medicine, the chemotherapy failure.Clinical data shows, although Gleevec can play persistent mitigation to CML, for the acute transformation phase patient, alleviates 2-6 after individual month, even it is also of no avail to continue administration, advancing of disease usually is accompanied by p210 ^BCR/ABLExpress and raise and the tyrosine kinase activity enhancing.

In sum, on the one hand, BCR-ABL specific inhibitor Gleevec has obtained remarkable curative effect clinically, shows that BCR-ABL is treatment CML and the good target of ALL really: promptly suppress the tyrosine kinase activity of BCR-Abl, and just can control disease; On the other hand, chemical sproof appearance has seriously hindered effective treatment of disease, and this just needs to seek the chemical sproof method that overcomes.The present invention is target spot with BCR-Abl, and designing the of new generation inhibitor different with the Gleevec combining site is the effective way of crossing over this obstacle, also is the effective way of exploring the leukemia targeted therapy.

The Bcr-Abl Tyrosylprotein kinase is at present unique being proved and the definite relevant target enzyme molecule of human leukemia morbidity, also is the desirable target of unique clear and definite antitumor drug.Gleevec is the tyrosine kinase inhibitor of first selectively targeted Bcr-Abl, clinical treatment chronic myelocytic leukemia, acute lymphoblastic leukemia and the gastrointestinal stromal tumors (GISTs) of being mainly used in.Yet when obtaining good efficacy, resistance also produces thereupon.Therefore developing new tyrosine kinase inhibitor will be the effective way that solves the resistance problem.This research is target spot with the disactivation conformation of ABL, use the new inhibitor that the screening of SGI medicine virtual screening workstation is different from the Gleevec binding site, obtained having the inside and outside to suppress active succinhydrazide compounds, and confirmed the restraining effect of this compound the BCR-Abl tyrosine kinase activity to sensitivity and drug-resistant leukemia model.

Summary of the invention

The objective of the invention is that (Gleevec, resistance STI-571) provide a kind of brand-new succinhydrazide compounds in order to overcome present imatinib.

Another object of the present invention has been to disclose the new preparation method of a kind of succinhydrazide compounds.

A further object of the present invention has provided the pharmaceutical composition that contains succinhydrazide compounds and one or more pharmaceutically acceptable carriers, vehicle or thinner composition.

Also purpose of the present invention has provided the application of succinhydrazide compounds in preparation treatment chronic myelocytic leukemia and adult's acute lymphoblastic leukemia medicine.

The invention provides compound with following formula I

A kind of preferable methods of preparation I compound is that wushu V and methyl iodide carry out grignard reaction and make compound VI, compound VI and hydrazine hydrate reaction are made compound VI I, make compound formula I with the paranitrobenzaldehyde condensation again, NMR and mass spectrum confirmation, fusing point are 230-232 ℃.

The synthetic route of hydrazide kind compound following (four steps)

The reaction of bromaniline and Succinic anhydried is normally finished by two kinds of reactants of equimolar amount, though other ratio also can be used.Reaction is preferably in a kind of non-proton inert solvent to be carried out, as benzene, toluene, acetonitrile, tetrahydrofuran (THF), chloroform, methylene dichloride or the like.Reaction is carried out under 0-100 ℃ of condition greatly, preferred 20-35 ℃.Compound V and iodomethane reaction are prepared compound VI, this reaction needs to carry out in inert solvent, and typical solvent is anhydrous diethyl ether, DMF, tetrahydrofuran (THF) or the like, and compound V is generally 1 with the mmol ratio of iodomethane reaction: 1-3.5, preferred 1: 1-1.2, about 24 hours of reaction times.Product VI is dissolved in C ₁-C ₄As methyl alcohol, ethanol or the like, add hydrazine hydrate in the lower alcohol, product VI is generally 1 with the mmol ratio of hydrazine hydrate: 10-30, preferred 1: 10-13, room temperature was placed one day, separated out white crystal, filtered to obtain product VII.With product VII and Ortho Nitro Benzaldehyde reflux stirring reaction, separate out white casse, filter and obtain product I, H-1NMR; C-12NMR proves conclusively compound, and fusing point is 230-232 ℃.

The pharmaceutical composition that contains the succinhydrazide compounds of the present invention can be prepared into various preparations with one or more pharmaceutically acceptable carriers, vehicle or thinner, comprises various solid orally ingestibles, liquid oral medicine, injection etc.

Solid dosage comprises tablet, discrete particles, capsule, slow releasing tablet, sustained release pellet or the like.Solid carrier can be at least a material, and it can serve as thinner, flavouring agent, solubilizing agent, lubricant, suspension agent, tackiness agent, disintegrating agent and coating agent.Inert solid carrier comprises trimagnesium phosphate, Magnesium Stearate, smoothers sugar, lactose, pectin, propylene glycol, Polysorbate 80, dextrin, starch, gelatin, cellulose substances for example methylcellulose gum, Microcrystalline Cellulose, low melt point paraffin, polyoxyethylene glycol, N.F,USP MANNITOL, theobroma oil etc.Liquid dosage form comprises solvent, suspension for example injection, infusion solution, powder preparation or the like.

The all right injection form administration of succinhydrazide compound of the present invention is though dosage changes according to treatment target, administering mode, symptom and other factor.When parenterai administration, composition of the present invention is made into injection formulations.Compound of the present invention is effective in quite wide dosage range.For example the dosage of taking every day can be in the scope of the about 0.1mg-500mg of per kilogram of body weight.In adult's treatment, dosage range once or is several times taken preferably at 1mg/kg--50mg/kg.The actual dosage of taking compound should be decided according to relevant situation by the doctor, these situations comprise by curer's physical state, the person's of choosing route of administration, age, body weight, patient are to the individual reaction of medicine, severity of patient's symptom or the like, therefore above-mentioned dosage range is not to limit the scope of the invention by any way.

Below in conjunction with in the body, anticancer experiment in vitro further sets forth positively effect of the present invention:

Succinhydrazide compounds among the present invention has biological activitys dose-dependently, this compound antagonism of method validation Gleevec drug-resistant leukemia model K562/G01 cells such as Western hybridization and co-immunoprecipitation to human erythroleukemia K562 and Gleevec resistance model K562/G01 cell.Seek antagonism Gleevec type leukemia cell and do not produce drug-fast new drug, explore the effective way of leukemia targeted therapy.

One, extracorporeal anti-tumor cell proliferation experiment:

Method: adopt conventional mtt assay to measure the medicine anti tumor activity in vitro, the tumour cell in the vegetative period of taking the logarithm is made into 1 * 10 with the RMPI RPMI-1640 that contains 10% calf serum ⁵/ ml, (cell count is 2 * 10 to be inoculated in the little culture plate in 96 holes ⁴/ hole, 180 μ l/ holes), at 37 ℃, 5%CO ₂Cultivate 24hr under the condition, the grouping dosing, each concentration is established three parallel holes, the treatment group adds the medicine of different concns, and negative control adds isopyknic physiological saline, and making final volume is 200 μ l/ holes, after cultivating 68hr, every hole adds MTT20 μ l (5mg/ml), and 37 ℃ are continued to cultivate 4hr, centrifugal (1000 rev/mins, 10min), abandon supernatant, every hole adds DMSO 150 μ l, and vibration is to precipitation dissolving fully; On microplate reader, detect 546nm optical density(OD) (OD) value.

The growth of tumour cell inhibiting rate calculates as follows:

Inhibiting rate (%)=[control group OD value-dosing group OD value]/control group OD value * 100%

Mapping can obtain dose response curve to the growth of tumour cell inhibiting rate with the different concns of same medicine, obtains the half casualty-producing concentrations IC50 of this medicine according to equation of linear regression, and promptly cell survival rate reduces by 50% o'clock drug dose.

The result: in order to seek the BCR/ABL inhibitor of new texture, we are that target spot carries out the screening of high-throughput computer with BCR/ABL.After 290,000 micromolecular compound three-dimensional structure databases are screened, obtain the brand-new succinhydrazide compounds of structure, it not only has lethal effect to responsive K562 cell, and maintenance is to the susceptibility of K562/G01 and K562/G02 mdr cell, Fig. 1 shows that this compound is respectively 32.39 ± 7.79 μ g/ml, 21.35 ± 7.90 μ g/ml and 32.86 ± 3.51 μ g/ml to the IC50 of K562 and K562/G01 cell.Prompting, this new compound not only can be killed the leukemia cell, and can overcome the resistance of tumour cell to Gleevec.Compound is to the influence of target protein (BCR-ABL) expression level:

Method: protein blot hybrid method (Western blot): the drug treating cell with PBS washing 2 times, added cold RIPA lysate after 5 hours on ice, with ultrasonic cell rupture of membranes instrument smudge cells, 4 ℃, 12000r/min, centrifugal 10min moves supernatant to another EP pipe.The cell pyrolysis liquid of getting the same protein amount carries out 12% polyacrylamide gel electrophoresis (SDS-PAGE), with the protein band electrotransfer of electrophoretic separation to nitrocellulose filter, after the antibody hybridization with anti-c-ABL, adding is connected with two of HRP accordingly and resists, and develops the color with DAB.

The result: Fig. 2 A as seen, formula I compound under 200 μ mol/L concentration, does not all produce obvious influence to the expression of BCR-ABL from 10 μ mol/L, pointing out this compound is to kill the leukemia cell's by the influence to kinase activity.

Compound is to the influence of target protein (BCR-ABL) kinase activity:

Principle: activatory ABL kinases can make self and the proteic tyrosine residues phosphorylation in downstream, and therefore, the amount of measuring phosphorylated tyrosine just demonstrates ABL kinase activity power indirectly.

Method: after the lysis with drug treating, in cell pyrolysis liquid, add c-ABL antibody, 4 ℃ of joltings are spent the night, add albumin A absorption immunocomplex, carry out the 12%SDS-PAGE electrophoresis, with the protein band electrotransfer of electrophoretic separation to nitrocellulose filter, respectively after the antibody hybridization with anti-actin and p-Tyr, adding is connected with two of HRP accordingly and resists, and develops the color with DAB.

The result: the succinhydrazide compounds is handled the K562 cell after 5 hours at 10 μ mol/L to 200 μ mol/L different concns, the proteic tyrosine of ABL is expressed the downward modulation (Fig. 2) that has in various degree in the total protein of cell extract, thereby shows that this compounds has certain restraining effect to the ABL tyrosine kinase activity.

The anti-tumor in vivo experiment:

Method: utilize the human leukemia K562 cell of BCR-ABL positive expression to set up the transplanted tumor in nude mice model, in order to assessing compound antitumor action in vivo.Balb/c nu/nu nude mice, female, 5 weeks of age week, body weight 12-17g, warp ¹³⁷Caesium 400rad irradiation, every mouse inoculation cell count was 1 * 10 in right fore root dorsal part subcutaneous vaccination K562 cell in the 3rd day ⁷Cell/0.2ml, the 5th day with the animal random packet, 3 every group.

Grouping and dosage setting: experiment minute negative control group and treatment group, negative control group gives corresponding solvent, and treatment group Chinese style I compound is established two groups of high dosage (750mg/Kg) and low dosages (500mg/Kg).In (being transplanted tumor inoculation back the 3rd day) beginning administration on random packet same day, at interval medication in 3 days once, continuous 5 times, abdominal injection.

Use three days one-shot measurement transplanted tumor in nude mice of vernier callipers model diameter of tumor, dynamic observe the anti-tumour effect of medicine, the calculation formula of gross tumor volume is:

V(mm ³)＝L×l ²/2

L and l are respectively the major diameter and the minor axis of tumour

Each relative tumour volume of measuring than (Relative Tumor Volume, calculation formula RTV) is:

RTV＝V _n/V ₀

V ₀The volume of measuring when beginning for GP TH, V _nVolume when measuring each time

The anti-tumor activity evaluation index is tumor control rate (IR%), and formula calculates below adopting:

IR％＝(1-T _RTV/C _RTV)×100％

T _RTVBe treatment group RTV, C _RTVBe control group RTV

Toxicity assessment: claim the nude mice body weight at every turn when measuring, calculate relative body weight than (Relative Body Weight is RBW) to assess the toxic reaction of medicine.

RBW＝W _n/W ₀

W _nBody weight during for measurement, W ₀Body weight when beginning for GP TH

The result: formula I compound exhibits goes out the anti-tumor in vivo activity of dose-dependently.Table 1 as seen, low dose compounds I (500mg/kg) treatment group tumor growth rate obviously slows down than the physiological saline control group, tumour inhibiting rate is 45.82mg/kg, and remarkable therapeutics meaning is arranged; The inhibitory rate to 63.81% of high dosage (750mg/kg), knurl volume and solvent control group have significant difference (P＜0.05) relatively, and from the therapeutics angle, this is significant, and formula I compound can effectively be controlled tumor growth in vivo.

When observing the antitumor drug drug action, must keep a close eye on the toxicity of compound itself, treatment back nude mice body weight change reflects the toxicity of medicine indirectly, we adopt relative body weight than the changing conditions of weighing nude mice body weight between different treatment groups.The analysis revealed of table 2 pair nude mice body weight is not treated the control group mice body weight and is rising tendency gradually, and 750mg/kg treatment group mouse body weight has downtrending, but with control group body weight there was no significant difference.500mg/kg treatment group body weight gain trend is suitable with control group.Show that this compound is safely and effectively under 750mg/kg and two therapeutic doses of 500mg/kg.

Table1 formula I compound is to the therapeutic action of nude mice K562 transplanted tumor

K562/A02 transplanted tumor group	Number of animals	Average knurl volume (mm 3)		RTV (Day21)	Tumour inhibiting rate (%) *	P value *
							Beginning	At last
		Control group 750mg/kg group 500mg/kg group	3 3 3						15.75 10.33 13.50	404.33 126 187.75	25.67 9.29 13.91	63.81 45.82	P＜0.05 P＜0.05

* compare with control group

Table2 formula I compound is to the influence of K562 transplanted tumor nude mice body weight

Group	Number of animals	Mean body weight (g)		RBW (Day16)	P value *
						Beginning	At last
		Control group 750mg/kg group 500mg/kg group	3 3 3					15.2 13.3 16.1	18.3 12.7 18.5	1.22 0.95 1.15	P＞0.05 P＞0.05

* compare with control group

Description of drawings:

Fig. 1 is the killing action of formula I compound to sensitivity and drug-resistant leukemia cell.Cell is measured the growth-inhibiting curve of medicine to human erythroleukemia K562 cell and Gleevce drug-resistant leukemia K562/G01, K562/G02 cell with mtt assay behind the formula I of different concns compound treatment 72h;

Fig. 2 is that formula I compound is to BCR-ABL tyrosine-kinase expression of enzymes and active influence.Lysing cell extracts total protein, with Western blotting method, and the influence that observation type I compound is expressed BCR-ABL under 200 μ M (A), 100 μ M (B), 50 μ M (C), 10 μ M (D); With the method for co-immunoprecipitation measure formula I compound under above-mentioned concentration to the influence of BCR-ABL kinase activity, (E) be solvent control;

Fig. 3 is the H-1NMR of formula I compound;

Fig. 4 is the C-12NMR of formula I compound.

Embodiment:

The present invention is described further below in conjunction with embodiment, embodiment only is indicative, mean that never it limits the scope of the invention by any way, compound of the present invention is through high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), fusing point (m.p.) detects, can adopt subsequently nucleus magnetic resonance ( ¹HNMR/ ¹³CNMR) etc. further prove conclusively its structure.

In the experiment needed intermediate can buy from market or can be easily method by top narration or other document known method make.

Embodiment 1

The preparation of mono succinate (to bromobenzene) acid amides (compound V)

With para-bromoaniline (7.08g, 40mmol) and Succinic anhydried (4.48g 40mmol) is dissolved in stirring at room in the 300ml chloroform, and reaction is spent the night, and separates out needle-like crystal, filters to obtain product V9.80g, productive rate 88.44%.

Embodiment 2

The preparation of succsinic acid 1-(to bromobenzene) acid amides 4-methyl esters (compound VI)

With product V (5g, 18mmol) and methyl iodide (3.33g 23mmol) is dissolved among the 100ml DMF, add saleratus (2.35g, 23mmol), stirring at room 16 hours. reaction solution dilutes with methylene dichloride, washing repeatedly, sodium bicarbonate is washed, anhydrous sodium sulfate drying.Steaming desolventizes and obtains product VI 5.0g, productive rate 95.45%.

Embodiment 3

The preparation of succsinic acid 1-(to bromobenzene) acid amides 4-hydrazides (compound VI I)

(38g 13mmol) is dissolved in the 150ml ethanol, and (0.72g, 144mmol), room temperature was placed one day, separated out white crystal, filtered and obtained product VII 3.5g, productive rate 87.72% to add hydrazine hydrate with product VI.

Embodiment 4

The preparation of succsinic acid 1-(to bromobenzene) acid amides 4-(m-nitro) acylhydrazone (Compound I)

With product VII (2.8g, 9.6mmol) and Ortho Nitro Benzaldehyde (1.6g 10.6mmol) is dissolved in the 100ml ethanol, and 100 ℃, the backflow stirring reaction is separated out white casse, filters to obtain product VII I3.8g, productive rate 90.48%.H-1NMR sees Fig. 3; C-12NMR sees Fig. 4.

In order to explain enforcement of the present invention more fully, provide following example of formulations.These embodiment explain rather than limit the scope of the invention.Preparation can adopt any one compound among the present invention as activeconstituents.

Embodiment 1

Every tablet preparation that contains the 100mg activeconstituents:

The mg/ sheet

Compound I 100

Lactose 50

Microcrystalline Cellulose 80

Starch 50

Hydroxyl methylcellulose 40

Magnesium Stearate 5

With activeconstituents, lactose, starch, Microcrystalline Cellulose are crossed 100 mesh sieves, and abundant mixing, the 2% hydroxyl methylcellulose aqueous solution joined in the above-mentioned mixed powder mix, cross 20 mesh sieve system softwoods, make wet granular in 45-55 ℃ of drying, carboxymethylstach sodium, Magnesium Stearate are joined compressing tablet in the above-mentioned dried particles.

Embodiment 2

Every capsule contains the capsular of 100mg activeconstituents and is prepared as follows:

Consumption/capsule weight concentration (%)

Compound I 100mg 30.0

Polyoxyethylene dehydration sorb 0.05mg 0.02

The sugar alcohol monoleate

Starch 250mg 69.98

Amount to 350.05mg 100.00

Embodiment 3

The preparation of injection

Compound I 100mg

Trisodium Citrate 50mg

PEG3000 10mg

Sodium hydroxide is an amount of

Distilled water 10ml

The pH value is filtered for 7.5-8.5, and filter liquor concentration is 1 mg/ml, and by 2 milliliters of packing of every peace bottle, sterilization promptly gets injection.

Claims (4)

1, the compound that has formula I

2, the application of the formula I compound of claim 1 aspect preparation treatment chronic myelocytic leukemia and Philadelphia chromosome male acute lymphoblastic leukemia medicine.

3, a kind of pharmaceutical composition that is used for the treatment of acute and chronic granulocyte leukemia, it comprises as the formula I compound of activeconstituents and one or more pharmaceutically acceptable carriers, vehicle or thinner.

4, the method for preparing following formula: compound:

It comprises the grignard prepared in reaction compound VI with formula V,

Compound VI and hydrazine hydrate prepared in reaction compound VI I;

Compound VI I and following formula: compound carry out prepared in reaction formula I compound

Images